Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism. Altered neocortica function involving changes at excitatory synapses likely underlie much of the cognitive and behavioral dysfunction in FXS and autism, and altered development of cortical network connectivity may be involved. Synapse elimination, or pruning, of glutamatergic synapses is hypothesized to be an important part of cortical network refinement during development. Pruning is thought to be impaired in FXS and autism - perhaps resulting in enhanced excitatory synaptic transmission and hyperexcitable networks. Support for normal pruning and its impairment in FXS comes mainly from measurements of spine morphology and electrophysiological responses in culture, but while these approaches have produced important advances, they have limitations. For example, the relationship between spines and actual synaptic function is unclear, and it is unclear how results obtained in culture relate to processes occurring in vivo. Therefore, large gaps in our knowledge of pruning in vivo and its impairment in FXS remain. We prepared acute mouse brain slices to obtain a snapshot of synaptic connectivity in vivo and used electrophysiological methods to examine cell-to- cell connections among cortical layer 5A (L5A) pyramidal neurons. We provide the first direct functional demonstration of pruning in the cortex by observing that cell-to-cell connections decline in number during a later developmental period - postnatal days 15 through 30 (P30). Unexpectedly, our results support a model where synapses are exclusively pruned at the level of cell-to-cell connections and not by individual synapses. In the FXS mouse - the Fmr1 knockout (KO) - connection pruning does not occur, and as a result, the network is hyperconnected at P30. We have also uncovered other developmental impairments in excitatory transmission in this cortical network. These results demonstrate that electrophysiological experiments examining this L5A network have great promise for an in depth examination of cortical refinement and pruning and could be employed for developing novel therapies that specifically correct impaired pruning in FXS. For this proposal, we address basic questions regarding altered pruning in the Fmr1 KO and develop techniques that would accelerate the study of synaptic developmental mechanisms in this model system. Our goal is to acquire preliminary data providing a stronger scientific and methodological rationale for a R01 proposal. We propose 2 optical stimulation methods in Aim 1 to both determine if increased connectivity with postsynaptic deletion of Fmr1 results in a net increase in excitation and determine if these approaches are effective for studying pruning mechanisms. In Aim 2, we propose virus infection experiments to determine the period at which FMRP loss-of-function causes hyperconnectivity. And in Aim 3, we determine the feasibility of studying this network in slice culture in order to complement the acute slice approach. This work would provide a foundation for studying alterations in synaptic formation or pruning in FXS and all related autism disorders.